A major area of interest in genome biology, especially in light of the determination of the complete nucleotide sequences of a number of genomes, is the targeted manipulation of genomic sequences. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U.S. Pat. No. 7,888,121 and U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; International Publication WO 2011/14612 (U.S. application Ser. No. 13/068,735) and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. See, also, Santiago et al. (2008) Proc Nat'l Acad Sci USA 105:5809-5814; Perez et al. (2008) Nat Biotechnol 26:808-816 (2008).
Artificial nucleases, which link the cleavage domain of a nuclease to a designed DNA-binding protein (e.g., zinc-finger protein (ZFP) or transcription activator like effector (TALE) linked to a nuclease cleavage domain such as from FokI), have been used for targeted cleavage in eukaryotic cells. For example, nuclease-mediated genome editing has been shown to modify the sequence of the human genome at a specific location by (1) creation of a double-strand break (DSB) in the genome of a living cell specifically at the target site for the desired modification, and by (2) allowing the natural mechanisms of DNA repair to “heal” this break. See, for example, U.S. Pat. No. 7,888,121 and U.S. application Ser. No. 13/068,735, the disclosures of which are incorporated by reference in their entireties for all purposes as well as U.S. Patent Publication Nos. 2011/0145940 and 2011/0201118.
To increase specificity, the cleavage event is induced using one or more pairs of custom-designed zinc finger nucleases that dimerize upon binding DNA to form a catalytically active nuclease complex. In addition, specificity has been further increased by using one or more pairs of nucleases that include engineered cleavage half-domains that cleave double-stranded DNA only upon formation of a heterodimer. See, e.g., U.S. Patent Publication Nos. 20080131962; 20090305346 and 20110201055, incorporated by reference herein in their entireties.
The double-stranded breaks (DSBs) created by artificial nucleases have been used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; 20070218528; 20070134796; 20080015164 and International Publication Nos. WO 07/014275 and WO 2007/139982 and U.S. Ser. No. 13/068,735, the disclosures of which are incorporated by reference in their entireties for all purposes. Thus, the ability to generate a DSB at a target genomic location allows for genomic editing of any genome.
There are two major and distinct pathways to repair DSBs—homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires the presence of a homologous sequence as a template (known as a “donor”) to guide the cellular repair process and the results of the repair are error-free and predictable. In the absence of a template (or “donor”) sequence for homologous recombination, the cell typically attempts to repair the DSB via the error-prone process of NHEJ.
Chromosomal translocations are chromosomal abnormalities wherein there is genetic rearrangement between non-homologous chromosomes. Found in 1 of every 625 newborns, these rearrangements are thought to be generally harmless but about 6% may play a role in human disease (see M. Oliver-Bonet; et al (October 2002). Molecular Human Reproduction 8 (10): 958-963, Brunet et al (2009) Proc. Natl. Acad. Sci., USA 106(26): 10620-10625). For example several cancers such as Burkitt's lymphoma, Mantle cell lymphoma, Follicular lymphoma, chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) and others are known to be associated with chromosomal translocations. In the case of CML and ALL, one chromosomal translocation that has been associated with these two diseases is the production of the so-called Philadelphia chromosome, which is a result of a reciprocal translocation between chromosome 9 and 22 wherein the translocation is designated t(9;22)(q34;q11). This particular translocation causes the unregulated activity of a tyrosine kinase. The tyrosine kinase inhibitor imatinib has been shown to have specificity for this tyrosine kinase and has proven to be a valuable tool for the treatment of CML.
However, there remains a need for additional methods and exogenous polynucleotides for creating targeted deletions at specific locations within the genome where the targeted deletions can range from small (e.g. a few base pairs) to large (e.g. hundreds of thousands of nucleotides) that can be used in numerous models, diagnostic and therapeutic systems. Also, there remains the need for additional models of specific chromosomal translocations to further develop novel therapeutics to treat diseases associated with these chromosomal abnormalities.